The objective of this study is to develop affinity reagents that react specifically with hemoglobin S at the 2,3-diphosphoglycerate (DPG) binding site and disrupt the sickling process. Recent work in my laboratory has led to the development of a class of such compounds for which the prototype is the bifunctional acylating agent bis (3.5-dibromosalicyl) fumarate. This compound reacts specifically with oxyhemoglobin to cross-link the beta chains of the tetramer. The cross-link bridge was shown by x-ray crystallographic studies to lie between Lys 82 beta1 an Lys 82 beta2, spanning the DPG site. This modification increases the solubility of deoxyhemoglobin S by approximately 35% which in vivo would have greatly beneficial therapeutic effects. Oxygen binding studies indicate that the cross-linked hemoglobin would be physiologically competent. Due to the lipophilic properties of the halogen substituents bis(3,5-dibromosalicyl) fumarate is able to cross the red cell membrane. We have shown in the mouse that the related compound bis(3.5-dibromosalicyl) succinate is able to modify hemoglobin in vivo and has very low toxicity. In the proposed research we will determine the effects of N-acylation of Lys 82 beta and Val 1 beta (the amino groups within the DPG site) by alternative cross-linking and also non-cross-linking reagents on the oxygen binding and solubility properties of hemoglobin S. This will allow us to extablish in vitro which modifications offer antisickling properties that may be clinically useful. Secondly we will identify those factors responsible for the highly selective modification of hemoglobin by bis(3,5-dibromosalicyl) fumarate and related affinity reagents, and we will determine the degree to which various lipophilic substituents increase the permeability of these reagents to the red cell membrane. These studies will be used as the basis for the design of new antisickling compounds in which the leaving group, the phenolic moiety of the acylating agent, is optimized with respect to both specificity and membrane permeability. Finally, the most promising antisickling reagents that are developed will be studied in suitable animals to determine their ability to modify hemoglobin in vivo and to assess their toxicity.